U.S. patent application number 11/055587 was filed with the patent office on 2007-11-22 for human derived monocyte attracting purified protein product useful in a method of treating infection and neoplasms in a human body, and the cloning of full length cdna thereof.
This patent application is currently assigned to THE UNITED STATES OF AMERICA, AS REPRESENTED BY. Invention is credited to Ettore Appella, Edward J. Leonard, Elizabeth A. Robinson, Teizo Yoshimura.
Application Number | 20070270329 11/055587 |
Document ID | / |
Family ID | 26973901 |
Filed Date | 2007-11-22 |
United States Patent
Application |
20070270329 |
Kind Code |
A1 |
Yoshimura; Teizo ; et
al. |
November 22, 2007 |
Human derived monocyte attracting purified protein product useful
in a method of treating infection and neoplasms in a human body,
and the cloning of full length cDNA thereof
Abstract
Pure peptide products, derived from either human glioma cell
line U-105MG or human peripheral blood mononuclear leukocytes are
provided; the products have a molecular mass of about 8,400
daltons, and the products exhibit optimal monocyte chemotactic
activity at a concentration of 1 nM. The cloning of full length
cDNA for the peptide products is also provided, as well as
recombinant methods for the production of monocyte chemoattractant
products. Methods of treating infection and neoplasms in a human
body with such peptides and monocyte chemoattractant products are
additionally provided, as well as pharmaceutical compositions for
the same.
Inventors: |
Yoshimura; Teizo;
(Frederick, MD) ; Robinson; Elizabeth A.;
(Bethesda, MD) ; Appella; Ettore; (Chevy Chase,
MD) ; Leonard; Edward J.; (Chevy Chase, MD) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
THE UNITED STATES OF AMERICA, AS
REPRESENTED BY
ROCKVILLE
MD
THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES
|
Family ID: |
26973901 |
Appl. No.: |
11/055587 |
Filed: |
February 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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07330446 |
Mar 30, 1989 |
6869924 |
|
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11055587 |
Feb 8, 2005 |
|
|
|
07304234 |
Jan 31, 1989 |
|
|
|
07330446 |
Mar 30, 1989 |
|
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Current U.S.
Class: |
514/2.3 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 31/00 20180101; C07K 14/523 20130101 |
Class at
Publication: |
514/002 |
International
Class: |
A61K 38/02 20060101
A61K038/02; A61P 31/00 20060101 A61P031/00 |
Claims
1-4. (canceled)
5. A method of treating infection in a human which comprises
administering to a human an effective infection treating amount of
the pure peptide product of claim 1.
6-19. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of copending
application Ser. No. 07/304,234 filed on Jan. 31, 1989.
BACKGROUND OF THE INVENTION
[0002] Macrophages play a central role in human immune responses
and defense against infection. Macrophages originate from blood
monocytes, which leave the circulation in response to several
signals that are thought in include chemoattractants elaborated at
foci of inflammation by tissue leukocytes stimulated by invading
microorganisms or by tissue injury. Heretofore, no pure, human
derived monocyte attracting substance has been provided.
SUMMARY OF THE INVENTION
[0003] The present invention is therefore concerned with providing
human derived, purified, products that exhibit monocytic
chemotactic activity (MCA). The invention is furthermore concerned
with the method utilized to isolate and purify these peptide
products, from human peripheral blood leukocytes and from a known
human glioma cell line, and with the cloning of monocyte
chemoattractant peptide full length cDNAs. The present invention is
also concerned with a method of treating infection and neoplasms in
a human body with monocyte chemoattractant peptide products
disclosed herein, and with pharmaceutical compositions for these
peptide products.
[0004] The present invention provides for:
[0005] A pure peptide product which may be derived from either (a)
human glioma cell line U-105MG, or (b) human peripheral blood
mononuclear leukocytes; said peptide product exhibiting optimal
monocyte chemotactic activity at a concentration of 1 nM; said
peptide product having an estimated molecular mass of about 8,400
daltons.
[0006] A pure peptide product, having a molecular mass of about
8,400 daltons, and exhibiting optimal monocyte chemotactic activity
at a concentration of 1 nM, said purified peptide obtained by the
process steps of:
[0007] (I) culturing live cells derived from: [0008] (a) human
glioma dell line U-105MG, or [0009] (b) human peripheral blood
mononuclear leukocytes, in an appropriate growth medium;
[0010] (II) separating said cells from said growth medium;
[0011] (III) chromatographing said growth medium on an Orange-A
Sepharose column, utilizing an appropriate solvent, and collecting
the fractions which contain the desired peptides;
[0012] (IV) chromatographing said peptide containing fractions
obtained in Step III on an appropriate cation-exchange HPLC column,
utilizing appropriate solvents, and collecting the fractions which
contain said desired peptides;
[0013] (V) chromatographing said peptide containing fractions
obtained in Step IV on a reverse phase HPLC column, utilizing an
appropriate solvent, and collecting the fractions containing said
desired peptides; and
[0014] (VI) removing liquids from said peptide containing fractions
obtained in Step V, to give said peptide product as a solid.
[0015] A method of preparing said purified peptide product, as
outlined in steps I-VI above.
[0016] A pure peptide product, derived from glioma cell line
U-105MG, said peptide product having an amino acid sequence of:
TABLE-US-00001 1 10 20 30 XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKE
40 50 60 70 AVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT
[0017] Wherein
[0018] A is alanine;
[0019] C is cysteine;
[0020] D is aspartic acid;
[0021] E is glutamic acid;
[0022] F is phenylalanine;
[0023] H is histidine;
[0024] I is isoleucine;
[0025] K is lysine;
[0026] L is leucine;
[0027] M is methionine;
[0028] N is asparagine;
[0029] P is proline;
[0030] Q is glutamine;
[0031] R is arginine;
[0032] S is serine;
[0033] T is threonine;
[0034] V is valine;
[0035] W is tryptophan;
[0036] Y is tyrosine; and
[0037] X is pyroglutamic acid.
[0038] A cDNA coding for a human monocyte chemoattractant
peptide.
[0039] A cDNA coding for a human monocyte chemoattractant peptide,
comprising the following nucleotide sequence, or a bioequivalent
thereof: TABLE-US-00002 CAG CCA GAT GCA ATC AAT GCC CCA GTC ACC TGC
TGT TAT AAC TTC ACC AAT AGG AAG ATC TCA GTG CAG AGG CTC GCG AGC TAT
AGA AGA ATC ACC AGC AGC AAG TGT CCC AAA GAA GCT GTG ATC TTC AAG ACC
ATT GTG GCC AAG GAG ATC TGT GCT GAC CCC AAG CAG AAG TGG GTT CAG GAT
TCC ATG GAC CAC CTG GAC AAG CAA ACC CAA ACT CCG AAG ACT;
wherein C is cytosine, T is thymine, A is adenine, and G is
guanine.
[0040] A cDNA coding for a human monocyte chemoattractant peptide,
which chemoattractant peptide comprises the following amino acid
sequence or a biological equivalent thereof: TABLE-US-00003 Gln Pro
Asp Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr Asn Arg Lys
Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr Ser Ser Lys Cys
Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala Lys Glu Ile Cys Ala
Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met Asp His Leu Asp Lys Gln
Thr Gln Thr Pro Lys Thr;
wherein,
[0041] Gly is glycine, THR is threonine, ASN is asparagine,
[0042] Ala is alanine, Pro is proline, Gln is glutamine,
[0043] Val is valine, Asp is aspartic acid, Cys is cystein,
[0044] Ile is isoleucine, Glu is glutamic acid, Met is
methionine,
[0045] Leu is leucine, Lys is lysine, Trp is tryptophan,
[0046] Ser is Serine, Arg is arginine, Phe is phenylalanine,
[0047] Tyr is tyrosine and His is histidine.
[0048] A method of treating infection in a human which method
comprises administering to the site of an infection in a human, an
effective infection treating amount of a purified peptide product,
either genetically engineered, or derived from either: (a) human
glioma cell line U-105MG, or (b) human peripheral blood mononuclear
leukocytes; said peptide product exhibiting optimal monocytic
chemotactic activity at a concentration of 1 nM; said peptide
product having a molecular mass of about 8,400 daltons.
[0049] A method of treating neoplasms in a human, which method
comprises administering to the site of a neoplasm in a human, an
effective neoplasm treating amount of a purified peptide product,
either genetically engineered, or derived from either (a) human
glioma cell line U-105MG, or (b) human peripheral blood mononuclear
leukocytes; said peptide product exhibiting optimal monocyte
chemotactic activity at a concentration of 1 nM; said peptide
product having a molecular mass of about 8,400 daltons.
[0050] A pharmaceutical composition comprising:
[0051] (I) a pure peptide product, either genetically engineered,
or derived from either: (a) human glioma cell line U-105MG, or (b)
human peripheral blood mononuclear leukocytes; said peptide product
exhibiting optimal monocyte chemotactic activity at a concentration
of 1 nM; said peptide product having an estimated molecular mass of
about 8,400 daltons; and
[0052] (II) a pharmaceutically acceptable carrier therefor.
[0053] The monocyte chemoattractant peptide of the present
invention has been purified to substantial homogeneity. Thus, the
term "pure" includes peptides which have been purified from various
sources by removal of contaminating human proteins and other
materials as well as peptides which have been synthesized or
produced in a substantially pure state by methods provided herein,
or by other methods. Preferably, the peptide of the present
invention is at least 98% free of other proteins and peptides.
[0054] The term "Glioma cell line U-105MG" refers to a human
derived cell line initiated by Pouten, J., and MacIntyre, E., "Long
term culture of normal and neoplastic gliomas", Acta Pathol.
Microbiol. Scand., Vol. 74, p. 465 (1968). The cell line has been
deposited with the American Type Culture Collection in Rockville,
Md. in accordance with the Budapest Treaty on deposits as Deposit
No. CRL 9932.
[0055] The terms "GDCF-1" and "GDCF-2" as used herein mean glioma
derived chemotactic factors 1 and 2.
[0056] The terms "LDCF-1" and LDCF-2" as used herein mean leukocyte
derived chemotactic factors 1 and 2.
[0057] The term "MCP" as used herein includes MCP-1 and mutants and
variants thereof, which are biologically equivalent to MCP-1. The
term also includes the monocyte chemoattractant peptides
hereinbefore labeled as GDCF-1, GDCF-2, LDCF-1 and LDCF-2, when the
same are genetically engineered.
[0058] The term "MCP-1 cDNA" as used herein means the cDNA sequence
illustrated in FIG. 2.
[0059] The term "MCP cDNA" as used herein means MCP-1 cDNA, and
biologically equivalent mutants and variants thereof, including
biologically active segments thereof.
[0060] The term "MCP-1" as used herein means human monocyte
chemoattractant protein-1 having the amino acid sequence
illustrated in FIG. 1.
[0061] The term "MCA" as used herein refers to monocyte chemotactic
activity as determined by an in vitro assay in a multiwell
chemotaxis chamber.
[0062] The terms "Unit of monocyte chemotactic activity" means the
reciprocal of dilution causing 50% of the maximal chemotactic
response.
[0063] The term "nM" as used herein means nanomole, i.e., 10.sup.-9
mole.
[0064] The term "MNL" as used herein means mononuclear
leukocyte.
[0065] The term "PHA" as used herein means phytohemagglutinin.
[0066] The term "appropriate growth medium" as used herein includes
RPMI 1640 medium containing 10% fetal calf serum.
[0067] The term "appropriate solvent" as used herein refers to
aqueous solutions of alkali earth metal salts, such as sodium
chloride and the like, when used in conjunction with
chromatographing on an Orange-A Sepharose column and
cation-exchange HPLC columns; and to organic solvent mixtures for
use with reverse phase HPLC columns.
[0068] The term "pharmaceutically acceptable carrier" as used
herein refers to conventional pharmaceutic excipients or additives
used in the pharmaceutical manufacturing art, and necessarily
includes while not limited to, those excipients or additives
contained herein under the caption "Pharmaceutical
Compositions".
[0069] The term "unit dosage form" as used herein refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
either a purified peptide product, MCP or MCP-1, calculated in an
amount sufficient to produce the desired effect in association with
a pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the novel unit dosage forms of the present
invention depend on the particular polypeptide employed and the
effect to be achieved, and the pharmacodynamics associated with
each compound in the host.
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] FIG. 1. A. Amino acid sequence of a portion of a peptide
fragment of a S. aureus VB protease digest of MCP-1. B. Probes
based on the above sequence. C. Structural organization and
sequencing strategy of human MCP-1 cDNA. Arrows show direction and
extend of determined sequences. Cross-hatched area indicates the
coding region for the mature form of MCP-1. Dotted region indicates
poly(A).
[0071] FIG. 2. Nucleotide sequence of human MCP-1. Triangle:
N-terminus of mature MCP-1. Dashed line: potential N-linked
glycosylation site. Solid line: sequence used for oligonucleotide
probe construction. Dotted line: polyadenylation signal.
[0072] FIG. 3. Expression of MCP-1 mRNA in tumor cell lines. Five
micrograms of poly(A) mRNA from each cell line were used. The first
5 blots are from glioma lines. SK-RC29, UMRC 2: renal cell
carcinomas. HOS: osteosarcoma. GCT: fibrous histiocytoma. HL60,
U937, Raji, Jurkat, H9: leukemia or lymphoma cell lines.
[0073] FIG. 4. Induction of MCP-1 mRNA in human PBMNL's by mitogens
or human recombinant cytokines. A. PBMNL's were cultured with 2.5
.mu.g/ml PHA or 10 .mu.g/ml LPS, and mRNA was extracted at the
indicated times. B. Cells were cultured with 100 U/ml of each
cytokine for 6 hours; then mRNA was extracted.
[0074] FIG. 5. Southern blotting analysis of human genomic DNA
digested with various endonucleases. A. EcoRI. B. BamHI. C. PstI.
D. HindIII.
[0075] FIG. 6. Hybridization of MCP-1 cDNA with genomic DNA from
various species.
DETAILED DESCRIPTION OF THE INVENTION
[0076] The following description is meant to aid those skilled in
the art in practicing the present invention. The examples which
follow should be considered as integral to this description, and
therefore it is to be regarded as advantageous that one practicing
the present invention, review the Examples contained herein in
conjunction with this detailed description. Furthermore, it is
noted that when one is practicing the present invention, or simply
reading the present disclosure, it should be understood that
certain terms such as peptide, polypeptide and protein can be used
interchangably, and as well that the terms MCP, MCP-1, GDCF-1,
GDCF-2, LDCF-1 and LDCF-2 can at times be used interchangably, such
as in recombinant synthesis methods provided for MCP and MCP-1
herein.
[0077] Methods for the isolation and purification of human monocyte
chemoattractant factor from human glioma cell line U-105MG is
provided in detail in Example (I) below, as is the isolation and
purification of monocyte chemoattractant factor from human
peripheral blood leukocytes in Example (II) below. Furthermore,
Example (III) below provides a detailed explanation as to the amino
acid sequencing of human monocyte chemoattractant factor. Example
(IV) below, provides for the cloning and coding of human monocyte
chemoattractant protein-1 cDNA (MCP-1 cDNA) which contains a gene
responsible for synthesis of human monocyte chemoattractant
factor.
[0078] Example (V) below, provides for the treatment of infection
with monocyte chemoattractant factor, and Example (VI) provides for
the treatment of neoplasts with monocyte chemoattractant
factor.
[0079] Example (VII) provides for a method of inhibiting the
actions of monocyte chemoattractant factor in vivo.
[0080] The degree of amino acid sequence homology with MCP-1 which
brings a protein within the scope of the definition of monocyte
chemoattractant protein (MCP) herein will vary depending upon
whether the homology between the candidate protein and MPC-1 falls
within or without the MCP-1 regions responsible for monocyte
chemoattractant activity; domains which are critical for monocyte
chemoattractant activity should exhibit a high degree of homology
in order to fall within the definition, while sequences not
involved in maintaining MPC-1 conformation or in effecting receptor
binding may show comparatively low homology. In addition, critical
domains may exhibit monocyte chemoattractant activity and yet
remain homologous as defined herein if residues containing
functionally similar amino acid side chains are substituted.
Functionally similar refers to dominant characteristics of the side
chains such as basic, neutral or acid, or the presence or absence
of steric bulk.
[0081] Generally, a protein defined as MCP will contain regions
substantially homologous with the FIG. 2 protein or fragments
thereof over a continuous block of from about at least 70 amino
acid residues, in particular the blocks encompassed by residues
23-99 in FIG. 2.
[0082] It is important to observe that any characteristics such as
molecular weight or the like, for the native or wild type mature
human MCP-1 of FIG. 2 obtained from peripheral lymphocyte or
established cell line cultures are descriptive only for the native
species of MCP-1. MCP, however, as contemplated by the definition
provided herein also includes other species which may not exhibit
all of the characteristics of native MCP-1. While MCP as defined
herein includes native MCP-1, other related proteins can fall
within the definition as well. For example, MCP-1 derivatives like
insertion mutants, deletion mutants, or fusion proteins may produce
MCP outside of a molecular weight established for native human
MCP-1 (fusion proteins with mature MCP-1 or MCP-1 itself as well as
insertion mutants will have a greater molecular weight than native,
mature MCP-1, while deletion mutants of native, mature MCP-1 will
have a lower molecular weight). Similarly, an MCP may be engineered
in order to reduce or eliminate susceptibility to hydrolysis by
trypsin or other proteases.
[0083] Note also that the language "biological equivalent" or
"bioequivalent" as used herein also includes MCP proteins which can
be converted, as by enzymatic hydrolysis, from an inactive state
analogous to a zymogen to a protein fragment which exhibits the
desired biological activity. Typically, inactive precursors will be
fusion proteins in which mature MCP-1 is linked by a peptide bond
at its carboxyl terminus to a human protein or fragment thereof.
The sequence at this peptide bond or nearby is selected so as to be
susceptible to proteolytic hydrolysis to release MCP or MCP-1,
either in vivo or, as part of a manufacturing protocol, in vitro.
MCP that is so generated then will exhibit monocyte chemoattractant
activity.
[0084] While MCP ordinarily is meant to mean human MCP, MCP from
sources such as other primates, or from such sources as murine,
porcine, equine or bovine is also considered included within the
definition of MCP above, so long as it meets the standards
described above for homologous regions and monocyte chemoattractant
activity.
[0085] MCP also includes multimeric forms, and multimers are
accordingly envisioned as suitable for use in in vivo therapy.
While it is thought desirable to express and recover MCP as a
substantially homogeneous multimer or monomer, MCP may be used
therapeutically as a mixture of different multimers.
[0086] Derivatives of MCP-1 are also included within the scope of
the term MCP. Such derivatives include, for example, amino acid
sequence mutants, glycosylation variants and covalent or
aggregative conjugates with other chemical moieties. Covalent
derivatives would generally be prepared by linkage of
functionalities to groups which are found in the MCP-1 amino acid
side chains or at the N- or C-termini, by means known in the art.
These derivatives may, for example, include: aliphatic esters or
amides of the carboxyl terminus or residues containing carboxyl
side chains, O-acyl derivatives of hydroxyl group-containing
residues, N-acyl derivatives of the amino terminal amino acid or
amino-group containing residues.
[0087] MCP-1 or MCP should preferably be synthesized in cultures of
recombinant organisms. Neither peripheral blood lymphocytes (PBLs)
nor cell lines are the most desirable (even though such are
utilized in Examples I and II herein). Since it is difficult in
practice to obtain PBLs of one class which are free of
contamination by cells of other classes, e.g. to obtain macrophages
free of B or T cells. Such contamination renders the separation
procedure applied to the products of such cells difficult because
of other potential protein release by contaminant cells.
Furthermore, MCP obtained from nonrecombinant culture is expensive
and consists solely of native MCP-1, such cultures thereby lacking
in the flexibility of recombinant culture to improve upon the
characteristics of MCP-1.
[0088] Alternatively, and preferably, MCP may be synthesized in
host cells transformed with vectors containing DNA encoding MCP-1
or more generally MCP. A vector is a replicable DNA construct.
Vectors may be used either to amplify DNA encoding MCP and/or to
express DNA which encodes MCP. An expression vector is a replicable
DNA construct in which a DNA sequence encoding MCP is operably
linked to suitable control sequences capable of effecting the
expression of MCP in a suitable host. Such control sequences
include a transcriptional promoter, an optional operator sequence
to control transcription, a sequence encoding suitable mRNA
ribosomal binding sites, and sequences which control termination of
transcription and translation.
[0089] DNA which encodes MCP-1 is obtained by chemical synthesis,
by screening reverse transcripts of mRNA from PBL (purified blood
leukocytes) or cell line cultures. Some suitable cell line for
culture are U-105MG, U-373MG and KMG-5.
[0090] This DNA is covalently labelled with a detectable substance
such as a fluorescent group, a radioactive atom or a
chemiluminescent group by methods known per se and including
fluorescent labeled probes as utilized in Example IV below. The DNA
is then used in conventional hybridization assays. Such assays are
employed in identifying appropriate MCP vectors and
transformants.
[0091] However, if one desires to culture MCP-1, without utilizing
recombinant DNA technology, then MCP synthesizing cells of U-105MG
(or other appropriate cell line) can be initially cultured in
conventional fashion until reaching a density of about
8-12.times.10.sup.5 cells/ml. The cells can then be transferred to
a serum-free medium and grown until a desired concentration of
MCP-1 has accumulated in the culture medium. Thereafter the culture
supernatant may be clarified by centrifugation or other means of
separating cell debris from the soluble components. Centrifugation
should be carried out at low speed so as to move only suspended
particles. The supernatant is then purified as described in either
Examples I or II below.
[0092] Suitable vectors comprise plasmids, viruses (including
phage), and integratable DNA fragments (i.e., integratable into the
host genome by recombination). Once it has transformed a suitable
host, the vector should replicate and function independently of the
host genome, or may, in some instances, integrate into the genome
itself. In the present specification, "vector" is generic to
"plasmid"; but plasmids are the most commonly used form of vector
at present. However, all other forms of vectors which serve an
equivalent function and which are, or become, known in the art are
suitable for use herein. Suitable vectors will contain replicon and
control sequences which are derived from species compatible with
the intended expression host. Transformed host cells are cells
which have been transformed or transfected with an MCP vector
constructed using recombinant DNA techniques. Transformed host
cells should ordinarily express MCP. Thus, the expressed MCP would
be deposited intracellularly or secreted into either the
periplasmic space or the culture supernatant, depending upon the
host cell selected.
[0093] DNA regions are operably linked when they are functionally
related to each other. For example, DNA for a presequence or
secretory leader is operably linked to DNA for a polypeptide if it
is expressed as a preprotein which participates in the secretion of
the polypeptide; a promoter is operably linked to a coding sequence
if it controls the transcription of the sequence; or a ribosome
binding site is operably linked to a coding sequence if it is
positioned so as to permit translation. Generally, operably linked
means continuous and, in the case of secretory leaders, contiguous
and in reading phase.
[0094] Suitable host cells are thought to be prokaryotes, yeast or
higher eukaryotic cells. Prokaryotes include gram negative or gram
positive organisms, for example, E. coli or Bacilli. Higher
eukaryotic cells also include established cell lines of mammalian
origin as described below. A preferred host cell could be
phage-resistant E. coli or M13 mp19, although other prokaryotes
could also be suitable.
[0095] Prokaryotic host-vector systems are also thought preferred
for the expression of MCP-1, and plethora of suitable microbial
vectors are available. Generally, a microbial vector would contain
an origin of replication recognized by the intended host, a
promoter which would function in the host and a phenotypic
selection gene, for example a gene encoding proteins conferring
antibiotic resistance or supplying an auxotrophic requirement.
Similar constructs could be manufactured for other hosts. For
example, E. coli is typically transformed using pBR322, a plasmid
derived from an E. coli species (Bolivar, et al., 1977, "Gene" 2:
95). pBR322 contains genes for ampicillin and tetracycline
resistance and thus provides easy means for identifying transformed
cells.
[0096] Vectors must contain a promoter which is recognized by the
host organism. This is generally a promoter homologous to the
intended host. Promoters most commonly used in recombinant DNA
construction include the .beta.-lactamase (penicillinase) and
lactose promoter systems (Chang et al., 1978, "Nature", 275: 615;
and Goeddel et al., 1979, "Nature", 281: 544), a tryptophan (trp)
promoter system (Goeddel et al., 1980, "Nucleic Acids Res." 8: 4057
and EPO App. Publ. No. 36,776) and the tac promoter [H. De Boer et
al., "Proc. Nat'l. Acad. Sci. U.S.A." 80: 21-25 (1983)]. While
these are the most commonly used, other known microbial promoters
could also be suitable.
[0097] MCP initially is recovered from cultures. Transformed
nonsecreting cells are lysed by sonication or other acceptable
method and debris separated by centrifugation, while the
supernatants from secreting cells (such as induced cell lines) are
simply separated from the cells by centrifugation.
[0098] Purification of monocyte chemoattractant from the
supernatant liquid can generally be had by purification methods
provided herein for purification of monocyte chemoattractant
peptide in Examples (I) and (II) wherein monocyte chemoattractant
is isolated and purified from cells.
[0099] The following Examples serve to further illustrate the
present invention; but the same should not be construed as limiting
to the scope of the invention disclosed herein.
EXAMPLE I
Purification of Monocyte Attracting Peptides from Human Glioma Cell
Line U-105MG
Materials and Methods
Cell Culture
[0100] Human glioma cell line U-105MG was utilized. Cells were
cultured in 150 cm.sup.2 tissue culture flasks (Costar, Cambridge,
Mass.) in RPMI 1640 medium (Advanced Biotechnologies Inc., Silver
Spring, Md.) supplemented with 10% fetal bovine serum (FBS,
HyClone, Logan, Utah), 20 mM L-glutamine and 50 .mu.g/ml
gentamycin. When cells became confluent, medium was replaced with
100 ml of FBS-free RPMI 1640 medium, which was collected 4 days
later and frozen at -20.degree. C.
Dye-Ligand Chromatography
[0101] Four liters of above obtained cultured fluid were
concentrated to 50 ml on a 150 mm diameter Amicon Diaflo membrane
(YM-5, molecular weight cutoff 5,000), dialyzed against 20 mM
tris-HCl, pH 8.0, and applied on a column of Orange-A Sepharose
(1.times.5 cm, Amicon Corp., Danvers, Mass.) that was equilibrated
with the same buffer. The column was eluted with a linear NaCl
gradient (limit 0.6M) at a flow rate of 0.5 ml/min; 2 ml fractions
were collected, and those with chemotactic activity were
pooled.
Cation Exchange HPLC
[0102] The pool of active fractions eluted from Orange-A Sepharose
was concentrated to 2 ml, dialyzed overnight at 4.degree. C.
against starting buffer (20 mM Mops, pH 6.5, in 0.1M NaCl) and
applied to a 0.75.times.7.5 cm CM 3SW column (Toyo Soda, Tokyo) at
room temperature. The column was eluted with a series of linear
NaCl gradients (limit 20 mM Mops, pH 6.5, in 0.4M NaCl) at a flow
rate of 1 ml/min. One ml fractions were collected and assayed for
chemotactic activity. Two separate peaks were found.
Reverse Phase HPLC
[0103] Each of the active peaks from the cation exchange column was
applied to a 0.5.times.25 cm Hi-Pore reverse phase column (BioRad,
Richmond, Calif.), equilibrated with a starting solvent of 0.1%
trifluoroacetic acid (TFA) in water. A linear gradient was
programmed, with a limit buffer of 70% (v/v) acetonitrile in water
containing 0.1% TFA. Flow rate was 1 ml/min; 1.0 ml fractions were
collected, and those in the region of A.sub.280 peaks were assayed
for chemotactic activity.
Results
Glioma Cell Line U-105MG Derived Peptides (GDCF-1 and GDCF-2)
[0104] Four liters of conditioned medium from U-105MG cells were
concentrated to 50 ml, dialyzed against starting buffer and applied
to an Orange-A Sepharose column. The column was eluted with a
linear NaCl gradient. The bulk of the protein did not bind to the
column, and emerged directly in the first 27 fractions. Chemotactic
activity bound to the column and was eluted between 0.2M and 0.45M
NaCl. As shown in Table 1, MCA was separated from about 98% of the
conditioned medium protein, and recovery of chemotactic activity
was 78%. Pooled active fractions were concentrated to 2 ml and
applied to a CM-HPLC column. Chemotactic activity was recovered in
two separate peaks that coeluted with two major A.sub.280 peaks.
Sequential fractions corresponding to the two MCA peaks were
analyzed by SDS-PAGE. The first MCA peak (GDCF-1), which had
maximal chemotactic activity in fractions 36 and 37, showed a major
band with maximal intensity in these fractions. There was also a
narrower band immediately about the major band, which could be seen
in the lanes of fractions 35 and 36. The second MCA peak (GDCF-2),
with maximal chemotactic activity in fractions 45 and 46, showed a
single major band with peak intensity in these fractions. By
reference to the mobility of protein standards, estimates of the
molecular masses of GDCF-1 and -2 were 15 kDa and 13 kDa. For
further purification, GDCF-1 (fraction 37) and GDCF-2 (fractions 45
and 46) were applied to reverse phase HPLC columns and eluted with
a linear acetonitrile gradient. Each MCA peak coeluted with a
single, sharp, A.sub.226 peak. The presence, in the chromatograms
of absorbance peaks without chemotactic activity showed that the
reverse phase column removed residual extraneous protein. This is
also shown in Table 1 by the increased specific activity of the
RP-HPLC products. When RP-HPLC GDCF-1 and GDCF-2 were analyzed by
SDS-PAGE, single bands were found, with estimated molecular masses
of 15 kDa and 13 kDa, respectively. As summarized in Table 1, from
4 liters of conditioned medium, about 5 .mu.g of GDCF-1 and 19
.mu.g of GDCF-2 were purified to apparent homogeneity. Specific
activity was 165 times that of the starting material for GDCF-1,
and 150 times for GDCF-2. Total recovery was approximately 13%.
Amino Acid Analysis of GDCF-1 and GDCF-2
[0105] Table 2 shows the amino acid composition of purified GDCF-1
and -2, based on two separate analyses of each peptide. Within the
limits of error of the method, the amino acid composition of the
peptides is identical. A minimal molecular mass, calculated from
the amino acid composition, is approximately 8400 daltons.
[0106] When N-terminal amino acid analysis was attempted, no
degradation of either peptide occurred, suggesting that the
N-terminus was blocked.
Assay of GDCF Chemotactic Activity for Monocytes and
Neutrophils
[0107] For both peptides, about 35% of monocytes added to assay
wells migrated at the optimal concentration of 1 nM. No significant
neutrophil migration was observed over a GDCF concentration range
of 0.01 to 30 nM in that experiment. Thus, showing GDCF attracts
monocytes but not neutrophils.
Assay to Distinguish Chemotaxis from Chemokinesis
[0108] Purified GDCF was added in different concentrations to top
and bottom wells of multiwell chambers, as outlined in Table 3.
Dose-dependent monocyte migration was observed only when GDCF was
in bottom wells. No significant migration occurred when top and
bottom wells contained equal concentrations of GDCF, showing that
migration was due primarily to chemotaxis, not chemokinesis.
Discussion of Results
[0109] Two chemotactic peptides for human monocytes, GDCF-1 and
GDCF-2, were purified to apparent homogeneity from culture fluid of
a human glioma cell line. Although these two peptides were
separated into two completely distinct peaks by CM-HPLC
chromatography, their elution patterns from a reverse phase HPLC
column were identical; and their amino acid compositions were
indistinguishable. Chemotactic potency and efficacy of both
peptides were very similar (Table III); and both were chemotactic
for monocytes but not neutrophils. It is possible that the two
peptides differ only by post-translation modifications, such as
phosphorylation, glycosylation or degradation. Based on the amino
acid composition, our estimate of the molecular mass of GDCF is
8400 daltons, which is considerably less than the 15 and 13 kDa
values determined by SDS-PAGE for GDCF-1 and -2. Discrepancies
between molecular mass estimates obtained by these different
methods of biologically active peptides have been reported by
others, e.g., Richmond, A., et al., Embo. J., Vol. 7, p. 2025-33
(1988).
[0110] As shown in the last column of Table 2, purification of GDCF
to homogeneity was associated with only a 150-fold increase in
specific activity, which reflects the relatively high concentration
of GDCF in U-105MG glioma cell culture fluid. This is due to the
absence of fetal bovine serum in the medium, and also indicates
that GDCF represents a significant percentage of the proteins
secreted by the U-105MG cell line.
[0111] The amino acid composition of GDCF is different from other
cytokines that have been reported to be chemotactic for monocytes
including IL-1, TNF, GM-CSF, M-CSF and TGF-beta. GDCF is also
distinct from other cytokines produced by glioma cells, including
IL-1 and platelet-derived growth factor.
Summary of Results
[0112] Two chemoattractants for human monocytes were purified to
apparent homogeneity from the culture supernatant of a glioma cell
line (U-105MG) by sequential chromatography on Orange-A Sepharose,
an HPLC cation exchanger and a reverse phase HPLC column. On
SDS-PAGE gels under reducing or non-reducing conditions, the
molecular masses of the two peptides (GDCF-1 and GDCF-2) were 15
and 13 kDa, respectively. Amino acid composition of these molecules
was almost identical, and differed from other cytokines that have
been reported. The N-terminus of each peptide was apparently
blocked. When tested for chemotactic efficacy, the peptides
attracted approximately 30% of the monocytes added to chemotaxis
chambers, at the optimal concentration of 10.sup.-9M. The activity
was chemotactic rather than chemokinetic. In contrast to their
interaction with human monocytes, the pure peptides did not attract
neutrophils.
EXAMPLE II
Purification of Monocyte Attracting Peptides from Human Peripheral
Blood Leukocytes
Cell Culture
[0113] Human peripheral blood mononuclear leukocytes (MNL's) were
isolated by metrizoate/Ficoll (Accurate Chemical and Scientific
Corp., Westbury, N.Y.) density sedimentation of leukapheresis
preparations obtained by the Blood Bank, Clinical Center, NIH, from
healthy human donors. Cells were washed three times with isotonic
phosphate buffered saline and resuspended in RPMI 1640 culture
medium (Advanced Biotechnologies, Inc., Silver Spring, Md.)
supplemented with 2 mM glutamine and 50 .mu.g/ml gentamycin. Cells
were cultured at a concentration of 5.times.10.sup.6 cells per ml
in tissue culture flasks with 2.5 .mu.g/ml phytohemagglutin (PHA)
(Sigma, St. Louis, Mo.). After incubation for 24 to 40 hrs, cells
were harvested; cell-free conditioned medium was obtained by
centrifugation at 400.times.g for 10 min.
Dye-Ligand Affinity Chromatography
[0114] For large scale purification, 4 liters of PHA culture
supernatant were concentrated to about 40 ml on a 150 mm diameter
Amicon Diaflo YM-5 membrane (m.w. cutoff 5000), dialyzed against 20
mM tris-HCl, pH 8.0, and applied on a 1.times.5 cm column of
Orange-A Sepharose (Amicon Corp., Danvers, Mass.) equilibrated with
the same buffer. The column was eluted at a flow rate of 0.5 ml/min
with a linear NaCl gradient to a limit of 0.6M in the same buffer.
Fractions were collected and analyzed for monocytic chemotactic
activity--thus indicating presence of a desired peptide.
High Pressure Liquid Chromatography Gel Filtration
[0115] Fractions containing peptides from the previous step were
utilized, and HPLC gel filtration was performed at room temperature
on a 7.5.times.600 mm TSK-2000 column (Toyo Soda, Tokyo, Japan),
equilibrated with phosphate buffered saline, pH 7.4. Fractions of
0.5 ml were collected at a flow rate of 1 ml/min. The column was
calibrated with bovine serum albumin (BSA), ovalbumin (OVA),
chymotrypsinogen A, cytochrome c, and aprotinin. Fractions were
collected and analyzed for MCA properties.
HPLC Chromatofocusing
[0116] Chromatofocusing was performed on a Mono P HR5/20 FPLC
column (Pharmacia LKB Biotechnology Inc., Piscataway, N.J.). Two pH
ranges were chosen, pH 7-4 and 9-6. For the 7-4 pH gradient,
starting buffer was 25 mM bis-tris, pH 7.1, and the column was
eluted with 10% (v/v) Polybuffer 74, pH 4.0. For pH range 9-6, 25
mM diethanolamine, pH 9.5, and 10% (v/v) Polybuffer 96, pH 6.0 were
used. MCA obtained from 8 gel filtration runs on TSK-2000 was
concentrated to 5 ml, and a 2 ml aliquot was dialyzed against
starting buffer in a 3,500 mw cutoff dialysis bag (Spectrum Medical
Industries Inc., Los Angeles, Calif.) and applied on a Mono P
column. The column was eluted at a flow rate of 1 ml/min. Two ml
fractions were collected; pH and chemotactic activity were
determined.
Cation Exchange HPLC
[0117] The pool of fractions with chemotactic activity eluted from
Orange-A Sepharose was concentrated and dialyzed against starting
buffer (20 mM Mops, p 6.5, 0.1M NaCl), and applied on a
0.75.times.7.5 cm CM-3SW-column (Toyo Soda, Tokyo) at room
temperature. The limit buffer was 20 mM Mops, pH 6.5, 0.4M NaCl. A
series of linear gradients was programmed at a flow rate of 1.0
ml/min; 1.0 ml fractions were collected.
Reverse Phase HPLC
[0118] The pool of fractions eluted from the cation exchange column
was applied to a 0.5.times.25 cm Hi-Pore reverse phase column
(Bio-Rad, Richmond, Calif.) equilibrated with a starting solvent of
0.1% trifluoroacetic acid in water. A linear gradient was
programmed, with a limit buffer of 70% (v/v) acetonitrile in water
containing 0.1% trifluoroacetic acid. Flow rate was 1.0 ml/min; 1.0
ml fractions were collected and assayed for MCA properties.
SDS Page
[0119] Electrophoresis was carried out on a vertical slab gel of
15% acrylamide with a discontinuous tris glycine buffer system.
Samples, as well as a solution of molecular weight standards, were
mixed with equal volumes of double strength sample buffer (20%
glycerol, 6% 2-mercaptoethanol), boiled, and applied to the gel.
After electrophoresis at 12 mA for 3 hrs, the gel was stained with
a silver staining kit (ICN Biomedicals, Irvine, Calif.).
Amino Acid Composition and Sequence Analysis
[0120] After a 24 hr hydrolysis in 6 M HCl in vacuo at 106.degree.
C., amino acid composition was determined on a Beckman System 6300
(Beckman Instruments, Fullerton, Calif.). N-terminal sequence
analysis was performed on an Applied Biosystems 470A Protein
Sequencer (Applied Biosystems, Foster City, Calif.).
Chemotaxis Assay
[0121] Mononuclear cells from human venous blood were separated by
centrifugation on metrizoate/Ficoll and used for chemotaxis in
multiwell chambers. Cell suspensions were added to upper wells of
the chambers; they were separated from lower wells containing
chemoattractant by a 10 .mu.m thick polycarbonate membrane with 5
.mu.m diameter holes. The number of monocytes that migrated through
the holes to the attractant side of the membrane during a 90 min
incubation was counted with an image analyzer. Results were
expressed as the percentage of the input number of monocytes that
migrated per well for duplicate wells. The reference
chemoattractant fMet-Leu-Phe (Peninsula Laboratories, Belmont,
Calif.) was dissolved in ethanol at a concentration of 1 mM and
diluted for assay.
Results
Molecular Sieve Chromatography on an HPLC Column
[0122] One hundred ml of culture medium harvested 40 hr after
addition of PHA to human MNL's was concentrated to 2 ml, and 200
.mu.l was injected into a TASK-2000 column. Eluted fractions were
assayed for chemotactic activity at 1:10 and 1:50 dilutions. As
shown in FIG. 1, several peaks of chemotactic activity were
detected at 1:10 dilution. At a 1:50 dilution, a single peak was
seen, which represented about 40% of total applied activity. The
center of this peak corresponded to a molecular mass of 17 kDa.
HPLC Chromatofocusing
[0123] The active fractions (34-40) from 8 runs on TSK-2000 were
pooled and concentrated to 5 ml. Two ml aliquots of this material
were used for chromatofocusing runs on a Mono P column. When pH
range 7-4 was used, two major chemotactic activity peaks were seen,
one in the pass through fractions and one at an early stage of the
pH gradient. At pH range 9-6, a single broad activity peak was seen
at pH 9.4 to 7.8, which represented about 85% of the applied
activity. After the pH gradient was completed, an additional 15% of
activity was eluted by 2M NaCl in fraction 34.
Affinity Chromatography on Orange-A Sepharose
[0124] Since human glioma cell derived monocyte chemotactic factor
could bind to Orange-A Sepharose, the binding capacity of the
leukocyte derived factor was studied. All of the 17 kDa, high pI
chemotactic activity bound to Orange-A Sepharose, and was eluted by
0.5M NaCl.
Purification of the Basic 17 kDa Chemotactic Factor
[0125] Since the 17 kDa chemotactic factor in the culture
supernatant of PHA-stimulated leukocytes behaved similarly to GDCF
on TSK-2000, Mono P and Orange-A Sepharose, the purification of
this factor was attempted by the same procedures as those for
GDCF.
[0126] Four liters of PHA culture supernatant were concentrated to
about 40 ml, dialyzed against starting buffer, and applied to an
Orange-A Sepharose column. About 50% of the activity passed through
the column without binding. This was not due to overloading, since
activity was seen in very early fractions. The bound activity was
eluted by NaCl (Table IV). Active fractions (40-56) were pooled,
concentrated, dialyzed, and applied to a cation exchange column for
further purification. By CM-HPLC chromatography, MCA was separated
into two distinct peaks which were eluted in the middle of the NaCl
gradient. Each of these peaks (fraction 39+40, fraction 49+50) was
further purified on a RP-HPLC column. Each MCA peak coeluted with a
sharp A.sub.226 peak (fraction 40). The behavior of this leukocyte
derived chemotactic activity on Orange-A Sepharose, CM-HPLC and
RP-HPLC was very similar to that of GDCF. Therefore, the two
chemotactic peptides purified from glioma cells (GDCF-1 and GDCF-2)
and the two chemotactic peptides purified from PHA-stimulated MNL's
(LDCF-1 and LDCF-2) were analyzed on a single SDS-PAGE gel. The
migration positions of the two glioma-derived peptides were
identical to the migration positions of the two MNL-derived
peptides, suggesting that the chemotactic peptides from these
different cell sources were identical.
Amino Acid Analysis
[0127] Table V shows that the amino acid composition of the two
leukocyte-derived chemotactic peptides is almost identical. A
minimal molecular mass, calculated from the amino acid composition,
is approximately 8400 daltons. Within the limits of error of the
method, the amino acid composition of LDCF is identical to that
previously determined for GDCF.
[0128] When N-terminal amino acid analysis was attempted, no
degradation of either peptide occurred, suggesting that the
N-terminus was blocked.
Comparison of Chemotactic Activity for Monocytes and
Neutrophils
[0129] Both peptides induced peak responses at 10.sup.-9M, at which
about 30% of input cells migrated. The magnitude of the response to
the two peptides was about the same as to fMet-Leu-Phe. Over the
concentration range studied, neither peptide induced chemotaxis
responses by human neutrophils.
Discussion of Results
[0130] Two peptides with chemotactic activity for human monocytes
were purified to apparent homogeneity from PHA-stimulated MNL's.
The two peptides eluted from a CM-HPLC column in different peaks,
and by SDS-PAGE had molecular masses of 15 and 13 kDa. However,
they had identical elution patterns by reverse phase HPLC, similar
amino acid compositions, and both had an apparently blocked amino
terminus. These similarities suggest that the two peptides are
derived from the same gene and differ because of post-translational
modifications. Such modifications might account for not only the
different elution patterns of LDCF-1 and -2 on CM-HPLC, but also
the discrepancy between the molecular mass estimates by SDS-PAGE
and those calculated from amino acid composition, Tung, J., et al.,
Biochem. Biophys. Res. Comm., Vol. 42, p. 1117 (1971). Similar
discrepancies have been reported by others for peptides in the same
size range, Richmond, et al., supra. In addition, it appears that
the two derived leukocyte derived chemotactic peptide are
indistinguishable from the glioma cell line U-105MG chemotactic
peptides obtained herein in Example I.
EXAMPLE III
Amino Acid Sequencing of Glioma Cell Line U-150MG Derived Human
Leukocyte Attracting Peptide (GDCF-2)
Materials and Methods
[0131] Purification of GDCF.
[0132] Human glioma cell-derived monocyte chemotactic factors were
purified from culture fluid of U-105MG cells by sequential
chromatography on an Orange-A Sepharose column, a cation exchange
column and a reverse-phase column as in Example I.
[0133] Chemical Modification of GDCF-2.
[0134] GDCF-2 was fully reduced and carboxymethylated with
iodo[2.sup.-3H]acetic acid (Amersham, 131 Ci/mol) as described by
Robinson et al, J. Biol. Chem., Vol. 254, p. 11418-11430 (1979).
Radioactivity was measured with an Analytic 81 liquid scintillation
counter; .sup.3H was counted with an efficiency of 44%.
Carboxymethylated GDCF was succinylated in 4 M urea-0.4M bicene, pH
8.6 with a 100-fold excess (over amino groups) of succinic
anhydride (Eastman).
[0135] Enzymatic Digestion and Peptide Purification.
[0136] Carboxymethylated GDCF-2 and a control peptide, Big Gastrin
1 (Sigma) with N-terminal pyroglutamic acid, were digested with
calf liver pyroglutamate aminopeptidase (Boehringer-Mannheim)
essentially as described by Podell, D., et al., Biochem. Biophys.
Res. Comm., Vol. 81, p. 176-85 (1978). Carboxymethylated GDCF-2 was
digested at 37.degree. C. in 50 mM NH.sub.4HCO.sub.3 with S. aureus
protease V8 (Boehringer-Mannheim, 1/25, w/w) for 6 h, and with
endoproteinase Asp-N (Boehringer-Mannheim 1/80, w/w) for 20 hr.
Carboxymethylated, succinylated GDCF-2 was digested at 25.degree.
C. with trypsin (Worthington 1/50 w/w) for 20 hr.
[0137] Each digest was subjected to automated Edman degradation (as
a mixture) before fractionation by HPLC. Peptides were purified by
HPLC using a Hewlett Packard 1090A Liquid Chromatograph and
Ultrapore RPSC C-3 or C-8 columns (Beckman) or an Applied
Biosystems Model 130A and an RP300 Aquapore column (Applied
Biosystems). Solvents were 0.10% trifluoroacetic acid in water (A)
and acetonitrile (B), respectively.
[0138] Amino Acid Composition and Edman Degradation.
[0139] Samples were hydrolyzed in vacuo in 6N HCl at 106.degree. C.
for 224 hr and analyzed on a Beckman System 6300. Edman degradation
was performed on an Applied Biosystems 470A equipped with an
on-line 120A PTH analyzer. PTH carboxymethylcysteine was detected
both by HPLC (eluting slightly earlier than PTH-Gln) and by
measurement of radioactivity (70 dpm .sup.3H/pmol Cys).
[0140] Mass Spectrometry.
[0141] Mass spectra was recorded on a tandem quadrupole Fourier
transform mass spectrometer constructed at the
[0142] University of Virginia. Operation of this instrument has
been described previously, Hunt, D., et al., Proc. Nat. Acad. Sci.
USA, Vol. 84, p. 620-23 (1987). Methodology for sequence analysis
of peptides by laser photodissociation on the Fourier transform
instrument has also been reported, Brinegar, A., et al., Proc. Nat.
Acad. Sci., USA, Vol. 85, p. 3927-31 (1988).
[0143] Samples for mass analysis on the tandem quadrupole Fourier
transform instrument were prepared by dissolving lyophilized HPLC
fractions in 2-10 .mu.l of 0.1% trifluoroacetic acid. A 0.5 to 1.0
.mu.l aliquot of these solutions (10-50 .mu.mol of peptide) was
added to 1 .mu.l of a 1/1 mixture of monothioglycerol/glycerol on a
gold-plated, stainless-steel probe tip, 2 mm in diameter. Peptides
were sputtered from this liquid matrix into the gas phase for mass
analysis largely in the form of (M+H).sup.+ ions by bombarding the
sample matrix with 6-10 keV Cs.sup.+ ion projectiles. The latter
ions were generated from a cesium ion gun (Antek, Palto, Calif.)
mounted directly on the ion source of the spectrometer.
[0144] Methyl Ester Formation.
[0145] A standard solution of 2 N HCl in methanol was prepared by
adding 800 .mu.l of acetyl chloride dropwise with stirring to 5 ml
of methanol. After the solution had stood at room temperature for 5
min, 100 .mu.l aliquots of the reagent were added to lyophilized
HPLC fractions. Esterification was allowed to proceed for 2 hr at
room temperature, and the solvent was then removed by
lyophilization.
Results
[0146] Edman degradation of GDCF-2 yielded no sequence data,
indicating that the N-terminus was blocked. Digestion with
pyroglutaminase did not remove the blocking group but removed
pyroglutamic acid from the control peptide. GDCF-2 was then
digested with endopeptidases. Sequence analysis of the products of
cleavage of carboxymethylated GDCF-2 with Staphylococcus aureus
protease V8 or carboxymethylated, succinylated GDCF-2 with trypsin
established the sequence of residues 19-76 (Table VI). The cleavage
at Ser-21 by protease V8 was unusual; however, the same cleavage
was observed in three separate digests. The sequence analysis data
are presented in Table VII.
[0147] Peptides TS1 (1-19) and SP1 (1-21) both had blocked
N-termini. SP1, containing the C-terminal sequence Arg-Lys-Ile-Ser,
was analyzed by mass spectrometry. A mass spectrum recorded on 20
pmol of this material showed an abundant (M+H).sup.+ ion at m/z
2454.3. Conversion of the oligopeptide to the corresponding methyl
ester shifted the observed (M+H).sup.+ ion to higher mass by 56
daltons, a result consistent with the addition of methyl groups
(mass 14) to two carboxymethyl Cys residues, a free C-terminus, and
one acidic residue in the peptide. Sub-digestion of SP1 fragment
with endo-Asp-N afforded a single large peptide, the mass spectrum
of which showed an abundant (M+H).sup.+ ion at m/z 2,246.2. Loss of
208 daltons in the above subdigestion can only be explained by
placing the residues, pGluPro, in positions one and two of the
parent molecule. Assignment of the third residue as Asp is dictated
by the specificity of the enzyme employed in the cleavage reaction.
An abundant fragment ion (m/z 2,131.3) resulting from the loss of
these three N-terminal residues on the mass spectrum of the parent
oligopeptide provided additional support for the above
assignment.
[0148] Additional sequence information at the C-terminus of the
endo-Asp-N cleavage product was obtained from fragmentation
observed in the mass spectrum of the product generated as a result
of on-probe acetylation. In this procedure the oligopeptide sample
dissolved in the thioglycerol/glycerol matrix is treated with a 3/1
methanol/acetic anhydride for 30 s and then inserted back into the
mass spectrometer. The resulting mass spectrum (M+H+=2331.4)
contained abundant fragment ions of the type Y'' at m/z 545, 659,
760, 907, 1021, and 1184 that allowed the C-terminal sequence to be
extended back from the C-terminus by an additional five residues.
This established the sequence of residues Tyr-13 to Ser-21.
[0149] Subdigestion of SP1 with both endo-Asp-N and chymotrypsin
afforded a single large oligopeptide, the methyl ester of which
afforded a mass spectrum containing an abundant ion at m/z 1342.1.
This is the predicted mass of the (M+H).sup.+ ion for the peptide
formed by cleavage of 8 residues from the C-terminus of the parent
molecule. The complete mass spectrum of this oligopeptide is shown
in FIG. 2. Fragment ions resulting from internal cleavage of the
chain at Pro-8 appear at m/z 197, 298, 473, 648, and 843, and allow
assignment of the sequence Pro-8 to Try-13. The last four of these
ions suffer partial loss of water and thus appear as doublets
separated by 18 mass units. Additional 18 mass unit doublets
corresponding to fragment ions of type Y'' (8) (m/z 896.6/914.6,
1010.7/1028.7, 1123.9/1141.9) allow placement of three additional
residues, Ile-Asn-Ala on the N-terminal side of Pro-8. The first
two residues in the peptide are assigned as Asp-Ala to account for
the remaining mass of the molecule (200.1 daltons) and the expected
specificity of the endo-Asp-N enzyme.
[0150] The N-terminal sequence obtained by tandem mass spectrometry
was subsequently confirmed in part as follows. Cleavage of
.sup.3H-carboxymethylated GDCF-2 with endoproteinase Asp-N yielded
a 51 residue peptide containing all the radioactivity. Edman
degradation of this peptide, D1, yielded a sequence corresponding
to the sequence of residues 3-23 (Table VII).
Discussion of Results
[0151] The complete amino acid sequence of GDCF-2 was determined by
Edman degradation and tandem MS. Although the sequence of residues
19-76 was obtained with relative ease by fragmentation and Edman
degradation, the sequence of the blocked N-terminal 18 residues
posed a difficult problem. Pyroglutamic acid was suspected to be
the N-terminal residue but digestion with pyroglutamate
aminopeptidase did not deblock GDCF-2 (due to the presence of
proline at position 2). Tandem MS provided the sequence of peptide
SP1 (1-21) expending only picomole amounts of the peptide. In
addition, partial sequence data for native GDCF-1 were obtained by
this method. These data indicate that GDCF-2 and GDCF-1 are
virtually identical molecules but that the N-terminus of GDCF-1 may
contain an additional residue and/or a different N-terminal
posttranslational modification (data not shown). The four
half-cystines of GDCF-1 were found to participate in two disulfide
bridges, Cys-11 or Cys-12 to Cys-36 and Cys-11 or Cys-12 to Cys-52
(GDCF-2 numbering).
[0152] The molecular weight of GDCF-2, calculated from the amino
acid sequence, is 8700 kDa whereas both native and
carboxymethylated GDCF-2 migrate as 13 kDa species on
NaDoDSO.sub.4/PAGE gels. We have no explanation for this
discrepancy since no post-translational modifications, other than
the formation of pyroglutamic acid, were detected in the sequence
analyses. A similar discrepancy between predicted and observed
molecular weight was reported for the melanoma growth factor, MGSA,
which consists of 73 amino acids, but migrates as a 13 kDa species,
Richmond, A., et al, supra. Anomalous migrations on
NaDoDSO.sub.4/PAGE gels have been commonly observed for basic
proteins, Tung et al., supra.
EXAMPLE IV
Cloning of Monocyte-Chemoattractant Protein-1 (MPC-1) Full Length
cDNA
[0153] In Example I above, we purified to homogeneity two human
monocyte chemoattractants from the culture fluid of a glioma cell
line. Although these two attractants could be separated into two
peaks by action exchange HPLC, their amino acid compositions were
identical. Likewise, two cation exchange HPLC peaks of monocyte
chemotactic activity, purified from culture fluid of PHA-stimulated
human blood lymphocytes in Example II, were indistinguishable in
amino acid composition to one another and to the glioma-derived
proteins. The complete amino acid sequence of one of the monocyte
chemoattractants purified from glioma culture fluid was determined
on a set of partial digests by a combination of Edman degradation
and mass spectrometry in Example III. A single protein chain with a
blocked N-terminus (pyroglutamic acid) and a total of 76 residues
was identified (see Table VI), in the present example it is named
Monocyte Chemoattractant Protein-1 (MCP-1). In the present example
we provide for the cloning of MCP-1 full-length cDNA, its
hybridization to genomic DNA from other species, and detection of
MCP-1 mRNA in normal cells stimulated by mediators of
inflammation.
Materials and Methods
[0154] Restriction enzymes, DNA modifying enzymes, and reagents for
cDNA preparation were from Bethesda Research Laboratories,
Bethesda, Md. DNA sequencing reagents were from United States
Biochemicals. Radiochemicals were from Amersham Corp. or New
England Nuclear. Lambda ZAP II vector was from Stratagene (La
Jolla, Calif.). Cytokines were from Boehringer Mannheim.
[0155] Total RNA was isolated from glioma cell line U-105MG by the
guanidinium-isothiocynate method; and poly(A) RNA was isolated by
oligo(dt)-cellulose chromatography, T. Maniatis, et al., "Molecular
Cloning: A Laboratory Manual" (Cold Spring Harbor Lab., Cold Spring
Harbor, N.Y. (1982)) herein incorporated by reference. cDNA was
synthesized by a modification of the Gubler and Hoffman method
[Gene, vol. 25, p. 263-269 (1983) herein incorporated by reference]
and was used to prepare a library in lambda ZAP II vector by the
method of J. M. Short, et al., Nucleic Acids Res., vol. 16, pp.
7583-7600 (1988), herein incorporated by reference.
Oligodeoxynucleotides were synthesized by the phosphoramidite
method of S. P. Adams, et al., J. Am. Chem. Soc., vol. 105, pp.
661-663 (1983) herein incorporated by reference, and purified by
HPLC. Probes (FIG. 1B) were synthesized on the basis of the
sequence of a peptide fragment (SP-4, FIG. 1A) generated by
digestion of MCP-1 with S. aureus V8 protease. Approximately
5.times.10.sup.5 recombinant phage from the cDNA library were
screened by high-density plaque hybridization [by a utilization of
the methods of T. Maniatis, et al., supra, and W. D. Benton, et
al., Science vol. 196, pp. 180-182 (1977) herein incorporated by
reference] with a mixture of .sup.32P-labeled oligonucleotides
SP-4-A and SP-4-B (FIG. 1B). Hybridization to nitrocellulose
filters was carried out overnight at 45.degree. C. in a solution
containing 6.times. standard saline citrate (SSC),
5.times.Denhardt's solution, 0.05% sodium pyrophosphate, 1%
NaDodSO.sub.4, 100 .mu.g/ml heat-denatured, sheared, salmon sperm
DNA and 1.times.10.sup.6 dpm/ml probe. Filters were washed once
with 6.times.SSC, 0.1% NaDodSO.sub.4 at 45.degree. C. for 5 min,
three times at 35.degree. C. for 30 min, and were dried and exposed
overnight to XS-5 film (Kodak) with an intensifying screen at
-80.degree. C. Phagemids carried within lambda ZAP II recombinants
were rescued with helper phage by the method of J. M. Short, et
al., supra. cDNA inserts were subcloned into M13mp19 by the method
of C. Yanisch-Perron, et al., herein incorporated by reference, and
single strands were sequenced on field gradient gels [such as those
provided by W. Ansorge, et al., J. Biochem. Biophys. Meth., vol.
10, pp. 237-243 (1984) herein incorporated by reference] by the
dideoxynucleoside triphosphate chain termination method of F.
Sanger, et al., Proc. Nat. Acad. Sci. U.S.A., vol. 74, pp.
5463-5467 (1977) herein incorporated by reference. Sequence data
were compiled and analyzed with computer assistance by a method
similar to that of C. Queen, et al., Nucleic Acids Res., vol. 12,
pp. 581-599, herein incorporated by reference.
[0156] Human PBMNL's were stimulated with 2.5 .mu.g/ml of PHA, 10
.mu.g/ml LPS, or 100 units/ml of the following human recombinant
LPS-free cytokines: IL-1.beta., IL-2, TNF.alpha., IFN-lambda.
Northern blot analysis of poly(A) RNA was done by the
glyoxaldimethylsulfoxide method [T. Maniatis, et al., supra] in a
1% agarose gel with a probe of MCP-1 cDNA insert labeled with
[.alpha.-.sup.32]CTP by random priming similar to the method of A.
P. Feinburg, et al., Anal. Biochem., vol. 132, pp. 6-13 (1983)
herein incorporated by reference. Filters were hybridized at
42.degree. C. overnight in 50% formamide, 1 M NaCl,
5.times.Denhardt's solution, 1 mM EDTA, 0.1% sarkosyl, 100 .mu.g/ml
sheared-denatured salmon sperm DNA, 1.times.10.sup.6 dpm/ml probe
and 50 mM piperazine-N,N'-bis[2-ethanesulfonic acid], pH 7. Filters
were washed twice with 2.times.SSC, 0.1% NaDodSO.sub.4 at
37.degree. C. for 30 min and 0.1.times.SSC, 0.1% NaDodSO.sub.4 at
50.degree. C. for 30 min prior to autoradiographic exposure.
[0157] Southern blot analysis was performed as described by T.
Maniatis, et al., supra, in a 1% agarose gel with 10 .mu.g
restriction-enzyme-cleaved DNA per lane. Hybridization was as
described for library screening except that transfers were made to
nylon filters, hybridization temperature was 65.degree. C. and the
probe was .sup.32P-labeled MCP-1 cDNA. Filters were washed once in
the hybridization solution used for library screening at 65.degree.
C. for 1 hr, then twice in 0.1.times.SSC, 0.1% NaDodSO.sub.4 at
48.degree. C. for 30 min.
Results
[0158] A cDNA library was constructed with poly(A) RNA from the
human glioma cell line (U-105MG) in cloning vector lambda ZAP II.
Approximately 5.times.10.sup.5 recombinant phage were screened with
the oligonucleotide probes shown in FIG. 1B. Forty-eight positive
signals on duplicate filters were obtained (-0.01% abundance).
Fifteen clones were plaque purified and phagemid DNA was prepared.
By preliminary nucleotide sequence analysis, at least three clones
coded for MCP-1. The insert from the clone with the longest 5'
untranslated region was sequenced (FIGS. 1C and 2).
[0159] Based on the amino acid sequence of pure MCP-1 as determined
in Example III, the mature form of the protein starts with
glutamine at residue 24 (nucleotide 70) (see FIG. 2). The amino
acid sequence deduced from nucleotides 70 to 297 is identical to
the directly determined 76 residue sequence of pure MCP-1. The cDNA
sequence contains an in-frame methionine triplet 69 nucleotides
upstream from the triplet corresponding to the NH.sub.2-terminus of
MCP-1. Seven of the 9 residues in the methionine triplet region,
CCAGCATGA, match the sequence reported by M. Kozak, Cell, vol. 44,
pp. 283-292 (1986) to be optimal for translation initiation. The
length and hydrophobic character of the deduced amino acid sequence
from the methionine to the NH.sub.2-terminus of MCP-1 are typical
of a signal peptide according to the teachings of G. von Heijne,
Eur. J. Biochem., vol. 133, pp. 17-21 (1983). There is a single
consensus sequence for N-linked glycosylation targeting amino acid
38.
[0160] The A+T content of the 3' untranslated region (66%) is not
nearly as high as that found in some transiently expressed mRNA's,
G. Shaw, et al., Cell, vol. 46, pp. 659-667 (1986). Unlike a number
of genes encoding proteins related to the inflammatory response [D.
Caput, et al., Proc. Natl. Acad. Sci. U.S.A., vol. 83, pp.
1670-1674 (1986)], there is no 8-nucleotide sequence, TTATTTAT, in
the 3' untranslated region.
[0161] In a survey of 5 different glioma cell lines, it was
reported that all released chemotactic activity for human monocytes
[Kuratsu, et al., J. Natl. Cancer Inst., vol. 81, pp. 347-351
(1989), incorporated herein by reference]. It was therefore of
interest to probe these and other tumor cell lines for MCP-1 mRNA
message. FIG. 3 shows Northern blots with a cDNA probe for MCP-1.
The high and low mRNA, respectively, of gliomas U-105MG and KMG-5
correlates with observed levels of chemotactic activity produced by
these two lines as reported by Kuratsu, et al., supra. MCP-1 mRNA
was not detected in other human tumor cell lines.
[0162] Since PBMNL-derived MCP-1 was indistinguishable from
glioma-derived MCP-1, we did Northern blot analyses of mRNA from
PBMNL's stimulated with PHA. No mRNA was detected before
stimulation, but high levels of mRNA were detected 3 and 6 hours
after addition of PHA (FIG. 4A). Ten .mu.g/ml of LPS also induced
high mRNA levels in these cells. IL-1B induced MCP-1 mRNA, though
the level was less than for PHA (FIG. 4B). Induction of MCP-1 mRNA
by IL-2, TNF.alpha., or IFN-lambda was not detected.
[0163] To identify genomic DNA fragments carrying the gene for
MCP-1, human DNA restriction endonuclease digests were analyzed by
Southern blot (FIG. 5). After BamHI or HindIII digestion, a single
band was seen. PstI digestion gave 2 major bands, which is in
agreement with the fact there is a PstI restriction site in the
MCP-1 cDNA. The data show that there is a single MCP-1 gene. DNA
from different species was digested with EcoRI and hybridized to
the same probe (FIG. 6). Under conditions of high stringency,
hybridization occurred with DNA of chimpanzee, baboon and capuchin,
but not of other species.
Discussion
[0164] In view of the fact that MCP-1's from glioma cells and
mitogen-stimulated PBMNL's are indistinguishable, either glioma
cells or stimulated PBMNL's can provide mRNA for cDNA library
construction. We selected the glioma cell line, since it produced
the attractant constitutively. The cDNA clone derived from the
glioma cell library detected mRNA in both glioma cells and
PHA-stimulated PBMNL's. This is consistent with our observation
that the amino acid compositions of MCP-1's from the two sources
are identical.
[0165] Although MCP-1 mRNA was detected in several glioma cell
lines, no message mRNA was found in 9 cell lines representing other
types of tumors. Thus, expression of the MCP-1 gene is not a
property of all neoplastic cells.
[0166] The amino acid composition of a monocyte chemoattractant
produced by aortic smooth muscle cells of the baboon [A. J.
Valente, et al., Biochemistry, vol. 27, pp. 41624168 (1988), herein
incorporated by reference] is identical to that of MCP-1 as
determined in Example III herein. Hybridization of the MCP-1 cDNA
probe with baboon DNA (FIG. 6) is added evidence for the
relationship between MCP-1 and the smooth muscle product, and
indicates that both lymphocytes and vascular smooth muscle cells
can produce this attractant.
EXAMPLE V
Treatment of Infection in a Human
[0167] When an effective, infection treating amount of one of the
purified peptide products, prepared in either Examples I or II
above, or MCP-1 synthesized by methods provided herein, is
administered to a human, and to the site of an infected area in a
human, control of that infection is expected. The volume of the
infection treating peptide composition to be administered, and the
frequency of administration will be determined by the treating
physician.
EXAMPLE VI
Treatment of a Neoplasm in a Human
[0168] When an effective, neoplastic treating amount of one of the
purified peptide products, prepared in either Examples I or II
above, or MCP-1 synthesized by methods provided herein, is
administered to a human, and to the site of a neoplasm in a human,
control of the neoplasm is expected due to peptide induced
accumulation of monocytes at the site. The volume of the
neoplasm-treating peptide composition to be administered, and the
frequency of administration will be determined by the treating
physician.
EXAMPLE VII
Treatment of Inflammatory Disease with a Peptide Inhibitor
[0169] Since the structure of the GDCF-2 peptide provided herein is
now known, as well as MCP-1, it is possible to synthesize short
peptides reflecting partial sequences of the complete GDCF-2
peptide or MCP-1 protein. These synthesized peptides can be
screened to find one that binds to the monocyte receptor site
without stimulating a chemotactic response. If such a peptide is
found, it can be used in clinical trials to control symptoms in
human chronic inflammatory diseases that are characterized by
inappropriate monocyte infiltration. The volume of the
infection-treating peptide composition to be administered, and the
frequency of administration will be determined by the treating
physician. TABLE-US-00004 TABLE I Purification of Human GDCF
Specific Total protein, Total MCA, activity mg units.sup.3 units/mg
Crude supernatant 29.sup.1 .sup. 200,000 6,900 Concentrated and
29.sup.1 .sup. 190,000 6,600 dialyzed supernatant Orange-A
Sepharose 0.52.sup.1 148,000 288,000 CM-HPLC P-I (frs 36 + 37)
0.03.sup.1 21,600 720,000 P-II (frs 45 + 46) 0.03.sup.1 18,200
607,000 Reverse phase HPLC GDCF-1 0.005.sup.2 5,700 1,140,000
GDCF-2 0.019.sup.2 20,000 1,053,000 .sup.1Protein concentration was
determined by dye protein assay with bovine serum albumin as
standard. .sup.2Protein concentration was calculated from amino
acid composition. .sup.3MCA concentration of 1 unit/ml was defined
as the reciprocal of the dilution at which 50% of the maximal
chemotactic response was obtained.
[0170] TABLE-US-00005 TABLE II Amino Acid Composition of Human GDCF
Residues per molecule.sup.1 Amino Acid GDCF-1 GDCF-2.sup.2 Asp +
Asn 7.6 8.0 Thr 6.8 6.8 Ser 4.6 4.6 Glu + Gln 8.4 8.0 Pro 5.1 4.5
Gly 2.0 0.3 Ala 5.7 6.1 Val 4.7 4.5 Met 0.9 0.7 Ile 5.3 5.0 Leu 2.3
2.3 Tyr 1.8 1.8 Phe 2.1 2.0 His 1.2 0.9 Lys 8.6 9.1 Arg 4.0 3.6 Cys
.sup. ND.sup.3 .sup. 3.5.sup.4 Trp ND ND .sup.1The data were
calculated on the basis of a total of 74 residual/molecule.
.sup.2GDCF-2 was reduced and .sup.3H-carboxymethylated for
composition analysis. .sup.3ND: not determined.
.sup.4H-carboxymethylcysteine.
[0171] TABLE-US-00006 TABLE III Assay to distinguish chemotactic
from chemokinetic activity in Glioma Cell Line U-105MG Derived
Purified Peptide Products Concentration Concentration in bottom
wells (M) in top wells 0 4 .times. 10.sup.-11 2 .times. 10.sup.-10
10.sup.-9 (M) Monocyte migration, % of input cell number .+-. SEM
A: GDCF-1 0 1 .+-. 0.2 5 .+-. 0.9 22 .+-. 2.4 35 .+-. 0.7 4 .times.
10.sup.-11 1 .+-. 0.2 4 .+-. 0.5 15 .+-. 1.3 34 .+-. 4.6 2 .times.
10.sup.-10 2 .+-. 0.4 2 .+-. 0.3 3 .+-. 1.2 21 .+-. 4.2 10.sup.-9 1
.+-. 0.2 1 .+-. 0.1 1 .+-. 0.1 3 .+-. 0.2 B: GDCF-2 0 2 .+-. 0.2 12
.+-. 1.8 25 .+-. 6.2 27 .+-. 3.9 4 .times. 10.sup.-11 1 .+-. 0.1 5
.+-. 0.5 18 .+-. 0.6 26 .+-. 5.0 2 .times. 10.sup.-10 3 .+-. 0.5 2
.+-. 0.2 5 .+-. 0.6 24 .+-. 1.5 10.sup.-9 1 .+-. 0.1 2 .+-. 0.1 2
.+-. 0.1 4 .+-. 0.1
[0172] TABLE-US-00007 TABLE IV Amino Acid Composition of Human
LDCF-1 and -2 Residues per molecule Amino Acid LDCF-1 LDCF-2 Asp +
Asn 8.1 7.8 Thr 6.4 6.7 Ser 5.6 4.7 Glu + Gln 9.4 8.9 Pro 5.4 5.2
Gly 2.2 3.2 Ala 6.2 6.0 Val 4.8 4.9 Met 0.7 0.9 Ile 4.8 5.2 Leu 2.4
2.3 Tyr 1.6 1.5 Phe 1.9 2.1 His 1.2 1.2 Lys 8.0 8.4 Arg 3.7 3.7 Cys
.sup. ND.sup.a ND Trp ND ND .sup.aND: not determined.
[0173] TABLE-US-00008 TABLE V Purification of Monocyte Chemotactic
Peptides Total Specific protein, Total MCA activity mg units.sup.c
units/mg Crude supernatant 79.sup.a 300,000 3,800 Concentrated and
57.sup.a 162,000 2,800 dialyzed supernatant Orange-A Sepharose
Pass-through 55.sup.a 89,000 1,600 Bound 1.7.sup.a 106,000 62,000
CM-HPLC P-I 0.10.sup.a 30,000 300,000 P-II 0.28.sup.a 16,000 57,000
RP-HPLC LDCF-1 0.042.sup.b 20,000 480,000 LDCF-2 0.020.sup.b 10,000
500,000 .sup.aProtein concentration was determined by dye protein
assay with bovine serum albumin as standard. .sup.bProtein
concentration was calculated from amino acid composition.
.sup.cChemotactic activity of 1 unit/ml was defined as the
reciprocal of the dilution at which 50% of the maximal chemotactic
response was obtained.
[0174] TABLE-US-00009 TABLE VI Amino Acid Sequence for GDCF-2 Amino
acid sequence of GDCF-2 deduced from S. aureus protease V8 (SP) and
aspartylendopeptidase P. fragi protease (D) peptides and from
tryptic peptides of succinylated GDCF-2 (TS). , tandem MS;
.sub.--------, Edman degradation; ------, unsequenced portions of a
particular peptide. 1 10 20 30
XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKE 40 50 60 70
AVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT
-----SP3---------------SP4-----------
-------------TS4--------------------- ------------- where: A is
alanine; C is cysteine; D is aspartic acid; E is glutamic acid; F
is phenylalanine; H is histidine; I is isoleucine; K is lysine; L
is leucine; M is methionine; N is asparagine; P is proline; Q is
glutamine; R is arginine; S is serine; T is threonine; V is valine;
W is tryptophan; X is tyrosine; and X is pyroglutamic acid.
[0175] TABLE-US-00010 TABLE VII Peptide PTH Amino Acid
(yield).sup.+ Cycle D1 SP2 SP3 SP4 TS2 TS3 TS4 1 D (27) V (72) A
(1494) I (191) K (320) L (60) I (279) 2 A (26) Q (84) V (582) C
(185) I (281) A (124) T (110) 3 I (24) R (23) I (490) A (168) S
(173) S (102) S (124) 4 N (21) L (60) F (463) D (123) V (200) Y
(78) S (158) 5 A (23) A (72) K (447) P (131) Q (130) R (27) K (76)
6 P (24) S (61) T (228) K (93) R (68) R (12) C (62) 7 V (11) Y (41)
I (329) Q (101) P (53) 8 T (10) R (34) V (300) K (81) K (64) 9 C
(11) R (37) A (321) W (30) E (53) 10 C (12) I (42) K (285) V (59) A
(42) 11 Y (12) T (34) E (143) Q (74) V (30) 12 N (12) S (28) D (56)
I (34) 13 F (16) S (27) S (37) F (29) 14 T (6) K (11) M (33) K (29)
15 N (8) C (22) H (18) T (18) 16 R (6) P (19) L (31) I (17) 17 K
(2) K (7) D (23) V (15) 18 I (9) E (5) K (17) A (20) 19 S (3) Q
(27) K (16) 20 V (3) T (14) E (10) 21 Q (8) Q (19) I (10) 22 -- T
(11) C (13) 23 L (6) P (8) A (11) 24 K (5) D (7) 25 T (5) P (7) 26
K (4) 27 Q (4) 28 K (1) 29 W (2) 30 V (2) 31 Q (2) 32 D (4) 33 S
(1) 34 M (1) 35 -- 36 H (2) 37 L (1) .sup.+The yield at each cycle
is in pmoles. C = carboxymethylcysteine, K = succinylated
lysine.
Pharmaceutical Compositions
[0176] The purified peptide products of the present invention, as
well as MCP-1 or synthesized MCP encompassed by the present
invention, may be made into pharmaceutical compositions by
combination with appropriate pharmaceutically acceptable carriers
or diluents, and may be formulated into preparations in solid,
semi-solid, liquid or gaseous forms such as tablets, capsules,
powders, granules, ointments, solutions, suppositories, injections,
inhalants, and aerosols in the usual ways for their respective
route of administration. The following methods of administration
and excipients provided therewith, are merely exemplary of
available methods which may be employed to deliver the purified
peptide products of the present invention to the site of an antigen
challenge, or a neoplasm in a human, and they should in no way be
construed as limiting the present invention.
[0177] In pharmaceutical dosage forms, the monocyte chemotactic
compounds of the present invention may be used along or in
appropriate association, as well as in combination with other
pharmaceutically active compounds.
[0178] In the case of oral preparations, the purified peptide
products of the present invention, as well as MCP-1 or MCP, may be
used alone or in combination with appropriate additives to make
tablets, powders, granules or capsules, e.g., with conventional
additives such as lactose, mannitol, corn starch or potato starch;
with binders such as crystalline cellulose, cellulose derivatives,
acacia, corn starch, potato starch or sodium
carboxymethylcellulose; with lubricants such as talc or magnesium
stearate; and if desired, with diluents, buffering agents,
moistening agents, preservatives and flavoring agents.
[0179] Furthermore, the peptide products of the present invention,
as well as MCP or MCP-1, may be made into suppositories by mixing
with a variety of bases such as emulsifying bases or water-soluble
bases.
[0180] The purified peptide products of the present invention, as
well as MCP or MCP-1, may be formulated into preparations for
injections by dissolving, suspending or emulsifying them in an
aqueous or non-aqueous solvent, such as vegetable oil, synthetic
aliphatic acid glycerides, esters of higher aliphatic acids or
propylene glycol; and if desired, with conventional additives such
as solubilizers, isotonic agents, suspending agents, emulsifying
agents, stabilizers and preservatives.
[0181] In the cases of inhalations or aerosol preparations, the
purified peptide products of the present invention, as well as MCP
or MCP-1, may be in the form of a liquid or minute powder in an
aerosol container with gas or liquid spraying agents, and if
desired, together with conventional adjuvants such as humidifying
agents. They may also be formulated as pharmaceuticals for
non-pressurized preparations such as in a nebulizer or an
atomizer.
[0182] The amount of the purified peptide products of the present
invention, as well as MCP or MCP-1 encompassed by the present
invention, to be used varies according to the degree of infection
or the size and type of neoplasm encountered in a human. A suitable
dosage is envisioned at about 0.001-1.0 mg/kg body weight per day
for treatment of infection or neoplasms in a human. The preferred
dosage being that amount sufficient to effectively treat an
infection or neoplasm in a human.
[0183] A method of treatment utilizing the purified peptide
products of the present invention, as well as MCP or MCP-1
encompassed by the present invention, can also be had by oral
ingestion of one of the peptides of the present invention with a
pharmaceutically acceptable carrier.
[0184] Unit dosage forms for oral administration such as syrups,
elixirs, and suspensions wherein each dosage unit, e.g.,
teaspoonful, tablespoonful, contains a predetermined amount of the
purified peptide product of the present invention or MCP or MCP-1.
Inclusion of pharmaceutically acceptable excipients, are readily
known by those skilled in the art.
[0185] Parenteral administration of the purified peptide products
of the present invention, as well as MCP or MCP-1, can be had by
administration with a pharmaceutically acceptable carrier, such as
Sterile Water for Injection, USP, or by normal saline.
[0186] The purified peptide products of the present invention, as
well as MCP or MCP-1, can be administered rectally via a
suppository. The suppository can include vehicles such as cocoa
butter, carbowaxes and polyethylene glycols, which melt at body
temperature, yet are solidified at room temperature.
[0187] The purified peptide products of the present invention, as
well as MCP or MCP-1, can be utilized in aerosol formulation to be
administered via inhalation. The purified peptide products can be
formulated into pressurized aerosol containers together with
pharmaceutically acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0188] It is also recognized that a skilled practitioner in the art
may desire to modify the above modes of administration, in order to
more effectively deliver one of the purified peptide products, as
well as MCP or MCP-1, directly to the site of an infection or
neoplasm in a human body. Such modification and direct
administration of one of the purified peptides of the present
invention, MCP or MCP-1, is fully comprehended herein, and
encompassed by the present invention.
[0189] Furthermore, it is envisioned that an injectable
pharmacological composition of the peptide products of the present
invention, as well as MCP or MCP-1, to be administered directly to
the site of an infection or neoplasm, would contain a concentration
of the peptide(s), encompassed herein, that is anticipated to cause
monocyte accumulation at locally injected tissue sites of human
patients. This concentration is thought to be preferably not less
than 10.sup.-8M and not more than 10.sup.-6M.
[0190] Lastly, it is to be understood that the present invention is
only limited by the scope of the appended claims.
Sequence CWU 1
1
13 1 76 PRT Homo sapiens MISC_FEATURE (1)..(1) Xaa is pyroglutamic
acid 1 Xaa Pro Asp Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe
Thr 1 5 10 15 Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg
Arg Ile Thr 20 25 30 Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe
Lys Thr Ile Val Ala 35 40 45 Lys Glu Ile Cys Ala Asp Pro Lys Gln
Lys Trp Val Gln Asp Ser Met 50 55 60 Asp His Leu Asp Lys Gln Thr
Gln Thr Pro Lys Thr 65 70 75 2 228 DNA Homo sapiens 2 cagccagatg
caatcaatgc cccagtcacc tgctgttata acttcaccaa taggaagatc 60
tcagtgcaga ggctcgcgag ctatagaaga atcaccagca gcaagtgtcc caaagaagct
120 gtgatcttca agaccattgt ggccaaggag atctgtgctg accccaagca
gaagtgggtt 180 caggattcca tggaccacct ggacaagcaa acccaaactc cgaagact
228 3 76 PRT Homo sapiens 3 Gln Pro Asp Ala Ile Asn Ala Pro Val Thr
Cys Cys Tyr Asn Phe Thr 1 5 10 15 Asn Arg Lys Ile Ser Val Gln Arg
Leu Ala Ser Tyr Arg Arg Ile Thr 20 25 30 Ser Ser Lys Cys Pro Lys
Glu Ala Val Ile Phe Lys Thr Ile Val Ala 35 40 45 Lys Glu Ile Cys
Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met 50 55 60 Asp His
Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr 65 70 75 4 9 PRT Homo
sapiens 4 Met Asp His Leu Asp Lys Gln Thr Gln 1 5 5 29 DNA
Artificial Sequence Probe misc_feature (6)..(6) n is inosine 5
gtctgngtct gcttatccaa atgatccat 29 6 29 DNA Artificial Sequence
Probe 6 gtttgcgttt gtttgtctaa gtggtccat 29 7 29 DNA Artificial
Sequence Probe misc_feature (6)..(6) n is inosine misc_feature
(18)..(18) n is inosine 7 gtctgngtct gcttatcnag atgatccat 29 8 29
DNA Artificial Sequence Probe 8 gtttgcgttt gtttgtccag gtggtccat 29
9 739 DNA Homo sapiens 9 ctaacccaga aacatccaat tctcaaactg
aagctcgcac tctcgcctcc agcatgaaag 60 tctctgccgc ccttctgtgc
ctgctgctca tagcagccac cttcattccc caagggctcg 120 ctcagccaga
tgcaatcaat gccccagtca cctgctgtta taacttcacc aataggaaga 180
tctcagtgca gaggctcgcg agctatagaa gaatcaccag cagcaagtgt cccaaagaag
240 ctgtgatctt caagaccatt gtggccaagg agatctgtgc tgaccccaag
cagaagtggg 300 ttcaggattc catggaccac ctggacaagc aaacccaaac
tccgaagact tgaacactca 360 ctccacaacc caagaatctg cagctaactt
attttcccct agctttcccc agacaccctg 420 ttttatttta ttataatgaa
ttttgtttgt tgatgtgaaa cattatgcct taagtaatgt 480 taattcttat
ttaagttatt gatgttttaa gtttatcttt catggtacta gtgtttttta 540
gatacagaga cttggggaaa ttgcttttcc tcttgaacca cagttctacc cctgggatgt
600 tttgagggtc tttgcaagaa tcattaatac aaagaatttt ttttaacatt
ccaatgcatt 660 gctaaaatat tattgtggaa atgaatattt tgtaactatt
acaccaaata aatatatttt 720 tgtacaaaaa aaaaaaaaa 739 10 99 PRT Homo
sapiens 10 Met Lys Val Ser Ala Ala Leu Leu Cys Leu Leu Leu Ile Ala
Ala Thr 1 5 10 15 Phe Ile Pro Gln Gly Leu Ala Gln Pro Asp Ala Ile
Asn Ala Pro Val 20 25 30 Thr Cys Cys Tyr Asn Phe Thr Asn Arg Lys
Ile Ser Val Gln Arg Leu 35 40 45 Ala Ser Tyr Arg Arg Ile Thr Ser
Ser Lys Cys Pro Lys Glu Ala Val 50 55 60 Ile Phe Lys Thr Ile Val
Ala Lys Glu Ile Cys Ala Asp Pro Lys Gln 65 70 75 80 Lys Trp Val Gln
Asp Ser Met Asp His Leu Asp Lys Gln Thr Gln Thr 85 90 95 Pro Lys
Thr 11 4 PRT Human MCP-1 peptide 11 Arg Lys Ile Ser 1 12 9 DNA
Human MCP-1 oligonucleotide 12 ccagcatga 9 13 8 DNA Artificial
Sequence Oligonucleotide in the 3' untranslated region 13 ttatttat
8
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